Project Summary Electron cryo-microscopy and computer-based, 3D image reconstruction techniques are revolutionizing the way structures of large, complex biomacromolecular machines are studied. These methods provide keys to understanding how these machines function. Our goal is to determine virus structures reliably at the highest possible resolutions in the shortest amount of time. This will enhance our ability to understand how viruses cause a variety of dieases and may yield important clues about how best to develop anti-viral agents. Microscopy and image reconstruction each pose their own set of significant challenges, and development of novel software clearly represents a key to the success of our research efforts as well as that of others. We aim to substantially enhance the computational capabilities that comprise the heart of the 3D structure determination part of our cryo-reconstruction work. The resolutions achieved and structures solved in cryo-reconstruction are limited by many factors, not the least of which includes the numerical techniques employed. Hence, we will focus on algorithmic improvements that expand the range of problems that can be studied, make it possible to reach higher resolutions, and reduce time to solution. For example, we will extend the capabilities of our software to examine the non-icosahedral components of nominally icosahedral viruses as well as particles with lower symmetries. In addition to the improvements noted above, our software development interests include automation, parallel computing, and enhancements to usability. For many virus structures, especially large and/or asymmetric ones, image reconstruction is the rate limiting step. To reduce the time between image acquisition and structure determination and also narrow the gap between the resolutions that can be reached by novice and expert users, we will enhance AUTO3DEM, our automation system for intelligently integrating the multiple applications required in image reconstruction. This system also provides a test bed for evaluating new ideas and makes the software easily accessible to a wider range of users. The proposed work should take us closer to our goal of real-time image reconstruction at the microscope, or at least tightly coupled to data acquisition, to provide rapid feedback and quickly screen sample properties. Our studies are also heavily leveraged by close ties to colleagues in cryo-microscopy, image processing, and data visualization and interpretation, the acquisition of modern microscopes with support from NIH, UCSD, and the Agouron foundation, and interaction with the San Diego Supercomputer Center. All of our software will be made readily accessible to the electron microscopy community. PUBLIC HEALTH RELEVANCE: Relevance Viruses infect nearly every living organism on Earth, causing serious diseases and death in humans and other animals, and also greatly impact food sources often leading to famine as well as weakened economies. Knowledge of virus structure provides an important link to better understand how viruses replicate themselves in susceptible hosts. Computer-based 3D models generated from virus images recorded in transmission electron microscopes, provide an important, first glimpse about the molecular structures of viruses. As the time required to obtain these models often requires weeks or months of dedicated effort, an important goal of this proposal is to reduce this down to hours or minutes.